Makio Ogawa

Hemorrhage, impaired hematopoiesis, and lethality in mouse embryos carrying a targeted disruption of the Fli1 transcription factor.

ABSTRACT The Ets family of transcription factors have been suggested to function as key regulators of hematopoeisis. Here we describe aberrant hematopoeisis and hemorrhaging in mouse embryos homozygous for a targeted disruption in the Ets family member, Fli1. Mutant embryos are found to hemorrhage from the dorsal aorta to the lumen of the neural tube and ventricles of the brain (hematorrhachis) on embryonic day 11.0 (E11.0) and are dead by E12.5. Histological examinations and in situ hybridization reveal disorganization of columnar epithelium and the presence of hematomas within the neuroepithelium and disruption of the basement membrane lying between this and mesenchymal tissues, both of which express Fli1 at the time of hemorrhaging. Livers from mutant embryos contain few pronormoblasts and basophilic normoblasts and have drastically reduced numbers of colony forming cells. These defects occur with complete penetrance of phenotype regardless of the genetic background (inbred B6, hybrid 129/B6, or outbred CD1) or the targeted embryonic stem cell line used for the generation of knockout lines. Taken together, these results provide in vivo evidence for the role of Fli1 in the regulation of hematopoiesis and hemostasis.

Transplanted Human Cord Blood Cells Give Rise to Hepatocytes in Engrafted Mice

Abstract: Recent studies suggest that rodent hepatocytes may be derived from hematopoietic stem cells. In the current study, the potential hematopoietic origin of hepatocytes was addressed using xenogeneic transplantation of human cord blood cells. CD34+ or CD45+ human cord blood cells were transplanted into “conditioned” newborn NOD/SCID/β2‐microglobulinnull mice. At 4 to 5 months post‐transplantation, livers of the recipient mice were cryosectioned and examined for evidence of human hepatocyte engraftment using RT‐PCR to detect human albumin mRNA, immunohistochemistry to detect human hepatocytic proteins, and fluorescence in situ hybridization (FISH) to detect the presence of human centromeric DNA. Analysis of the bone marrow of transplanted mice revealed that 21.0–45.9% of the cells were human CD45+ cells. FISH analysis of frozen sections of transplanted mouse liver revealed the presence of engrafted cells positive for human centromeric DNA. That engrafted human cells functioned as hepatocytes was indicated by the expression of human albumin mRNA, as judged by RT‐PCR. FISH analysis with human and mouse centromeric DNA probes excluded spontaneous cell fusion as the cause for the generation of human hepatocytes. Human cord blood cells can give rise to hepatocytes in a xenogeneic transplantation model. This model will be useful to further characterize the cord blood progenitors of hepatocytes.

Developmental changes of CD34 expression by murine hematopoietic stem cells

It has been reported that fetal murine hematopoietic stem cells are CD34(+), whereas adult stem cells are CD34(-). We sought to delineate the developmental changes of CD34 expression by hematopoietic stem cells and carried out systematic analysis of long-term engrafting cells in the bone marrow and/or blood of perinatal, juvenile, and adult mice. To obtain information on the total population of stem cells, we prepared CD34(+) and CD34(-) populations of mononuclear cells without prior enrichment and assayed their long-term reconstituting abilities by transplantation into lethally irradiated Ly-5 congenic mice. All stem cells from perinatal to 5-week-old mice were CD34(+). In 7-week-old mice, CD34(-) stem cells began to emerge, and the majority of the stem cells were CD34(-) in the 10- and 20-week-old mice. Approximately 20% of adult stem cells expressed CD34. Developmental changes of CD34 expression from the positive to the negative state takes place between 7 and 10 weeks of age for the majority of murine stem cells. Approximately 20% of adult stem cells remain CD34(+). These observations provide insight into the current controversy regarding CD34 expression by adult hematopoietic stem cells and suggest that the majority of stem cells in human umbilical cord blood and bone marrow of young children are CD34(+).

Contribution of Bone Marrow Hematopoietic Stem Cells to Adult Mouse Inner Ear: Mesenchymal Cells and Fibrocytes

Bone marrow (BM)‐derived stem cells have shown plasticity with a capacity to differentiate into a variety of specialized cells. To test the hypothesis that some cells in the inner ear are derived from BM, we transplanted either isolated whole BM cells or clonally expanded hematopoietic stem cells (HSCs) prepared from transgenic mice expressing enhanced green fluorescent protein (EGFP) into irradiated adult mice. Isolated GFP+ BM cells were also transplanted into conditioned newborn mice derived from pregnant mice injected with busulfan (which ablates HSCs in the newborns). Quantification of GFP+ cells was performed 3–20 months after transplant. GFP+ cells were found in the inner ear with all transplant conditions. They were most abundant within the spiral ligament but were also found in other locations normally occupied by fibrocytes and mesenchymal cells. No GFP+ neurons or hair cells were observed in inner ears of transplanted mice. Dual immunofluorescence assays demonstrated that most of the GFP+ cells were negative for CD45, a macrophage and hematopoietic cell marker. A portion of the GFP+ cells in the spiral ligament expressed immunoreactive Na, K‐ATPase, or the Na‐K‐Cl transporter (NKCC), proteins used as markers for specialized ion transport fibrocytes. Phenotypic studies indicated that the GFP+ cells did not arise from fusion of donor cells with endogenous cells. This study provides the first evidence for the origin of inner ear cells from BM and more specifically from HSCs. The results suggest that mesenchymal cells, including fibrocytes in the adult inner ear, may be derived continuously from HSCs. J. Comp. Neurol. 496:187–201, 2006.

Reversible expression of CD34 by adult human bone marrow long-term engrafting hematopoietic stem cells.

OBJECTIVE We previously reported that CD34(-) population of bone marrow (BM) cells from adult humans contains cells capable of engraftment and multilineage differentiation. We also reported on the reversibility of CD34 expression by murine hematopoietic stem cells. Based on long-term observations in primary, secondary, and tertiary sheep recipients, we now present definitive evidence for the long-term engrafting capability of human BM CD34(-) cells, and the reversibility of CD34 expression by human BM hematopoietic stem cells (HSC) in vivo. MATERIALS AND METHODS We used serial transplantations into primary, secondary, and tertiary preimmune fetal sheep recipients to evaluate and compare the long-term engraftment and differentiation of adult human bone marrow-derived CD34(-) and CD34(+) cells in vivo. RESULTS In primary hosts CD34(-) or CD34(+) cells produced multilineage human cell activity that persisted for 31 months. To confirm the long-term engrafting characteristics of CD34(-) cells and determine whether CD34 expression on human HSC is reversible, we transplanted human CD34(-) and CD34(+) cells obtained from primary hosts into secondary sheep recipients. Multilineage engraftment occurred in all secondary hosts, and in tertiary hosts transplanted with CD34(-) or CD34(+) cells obtained from BM of secondary recipients. CONCLUSION These results demonstrate that human BM CD34(-) cells are capable of long-term multilineage engraftment in vivo. The finding that both CD34(-) and CD34(+) cells from primary/secondary groups engraft secondary/tertiary hosts indicates that CD34 expression on human HSC is reversible, a process that does not impair HSC function in vivo.

Changing phenotypes of hematopoietic stem cells

Identification of the surface phenotypes of hematopoietic stem cells is an important subject in both clinical stem cell transplantation and basic research in hematopoiesis. Transplantation of pure populations of stem cells should eliminate the occurrence of graft-vs-host disease (GVHD) in allogeneic transplantation and may reduce the recurrence rate of malignancies in autologous transplantation. Availability of highly enriched populations of stem cells should facilitate investigations in a number of areas such as in vitro expansion, cryogenic storage, and gene therapy using stem cells. An ultimate goal is to develop a method to quantitate the number of living stem cells based on their surface characteristics. During the last two decades, investigators identified a number of surface molecules, which, in combinations, were thought to provide exact definition of murine and human stem cells. Recent studies of murine stem cells, however, clearly demonstrated that expression of the surface antigens of stem cells is under the influence of developmental stages and the kinetic state of the stem cells. This review summarizes the concept of the changing phenotypes of hematopoietic stem cells.

Hematopoietic stem cell origin of adipocytes

OBJECTIVE It has generally been believed that adipocytes are derived from mesenchymal stem cells via fibroblasts. We recently reported that fibroblasts/myofibroblasts in a number of tissues and organs are derived from hematopoietic stem cells (HSCs). In the present study, we tested the hypothesis that HSCs also give rise to adipocytes. MATERIALS AND METHODS Using transplantation of a single enhanced green fluorescent protein-positive (EGFP(+)) HSC and primary culture, we examined generation of adipocytes from HSCs. RESULTS Adipose tissues from clonally engrafted mice showed EGFP(+) adipocytes that stained positive for leptin, perilipin, and fatty acid binding protein 4. A diet containing rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, significantly enhanced the number of EGFP(+) adipocytes. When EGFP(+) bone marrow cells from clonally engrafted mice were cultured under adipogenic conditions, all of the cultured cells stained positive with Oil Red O and Sudan Black B and exhibited the presence of abundant mRNA for adipocyte markers. Finally, clonal culture- and sorting-based studies of Mac-1 expression of hematopoietic progenitors suggested that adipocytes are derived from HSCs via progenitors for monocytes/macrophages. CONCLUSION Together, these studies clarify the current controversy regarding the ability of HSCs to give rise to adipocytes. Furthermore, our primary culture method that generates adipocytes from uncommitted hematopoietic cells should contribute to the studies of the mechanisms of early adipocytic differentiation and may lead to development of therapeutic solutions for many general obesity issues.

Defective Megakaryopoiesis and Abnormal Erythroid Development in Fli-1 Gene-Targeted Mice

Mouse embryos homozygous for a targeted disruption in theFli-1 gene show hemorrhage into the neural tube and brain on embryonic day (E)11.0 and die shortly thereafter. Livers from the mutant embryos contain drastically reduced numbers of pronormoblasts, basophilic normoblasts, and colony-forming cells.To determine the nature of impaired hematopoiesis, we carried out cell culture studies of mutant embryonic stem (ES) cells and cells from the aorta-gonad-mesonephros (AGM) region of E10.0 mutant embryos. There was a striking reduction in the number of megakaryocytes in cultures of mutant AGM cells compared with cultures of AGM cells from wild-type or heterozygous embryos. Furthermore,Fli-1 mutant ES cells failed to produce megakaryocyte colonies and multilineage colonies containing megakaryocytes. Consistent with the observed defect in megakaryopoiesis, we also demonstrated the down-regulation of c-mpl in the AGM of mutant embryos. The percentages of pronormoblasts and basophilic normoblasts were significantly reduced in cultures of mutant AGM embryos, which contained primarily polychromatophilic and orthochromatic normoblasts. These results provide further evidence for the role ofFli-1 in the regulation of hematopoiesis and for c-mpl as aFli-1 target gene.

Human cord blood long-term engrafting cells are CD34 + CD38 −

There have been controversies about CD34 and CD38 expression by human cord blood (CB) stem cells. Using the newborn NOD/SCID/β2-microglobulin-null mouse assay that we recently developed, we examined the in vivo engrafting capability of human CB cells. Almost all of the 4–5 months engrafting cells were found in CD34+ population. The capability of secondary reconstitution was found only in the CD34+ cells. When the CD34+ CB cells were separated into CD38− and CD38+ subpopulations and tested for engraftment, the majority of the engrafting cells were detected in the CD38− subpopulation. These findings are consistent with the results from studies of murine stem cells and strongly indicate that the phenotype of human CB stem cells is CD34+ CD38−.

Amelioration of a mouse model of osteogenesis imperfecta with hematopoietic stem cell transplantation: Microcomputed tomography studies

OBJECTIVE To test the hypothesis that hematopoietic stem cells (HSCs) generate bone cells using bone marrow (BM) cell transplantation in a mouse model of osteogenesis imperfecta (OI). OI is a genetic disorder resulting from abnormal amount and/or structure of type I collagen and is characterized by osteopenia, fragile bones, and skeletal deformities. Homozygous OI murine mice (oim; B6C3Fe a/a-Col1a2(oim)/J) offer excellent recipients for transplantation of normal HSCs, because fast turnover of osteoprogenitors has been shown. MATERIALS AND METHODS We transplanted BM mononuclear cells or 50 BM cells highly enriched for HSCs from transgenic enhanced green fluorescent protein mice into irradiated oim mice and analyzed changes in bone parameters using longitudinal microcomputed tomography. RESULTS Dramatic improvements were observed in three-dimensional microcomputed tomography images of these bones 3 to 6 months post-transplantation when the mice showed high levels of hematopoietic engraftment. Histomorphometric assessment of the bone parameters, such as trabecular structure and cortical width, supported observations from three-dimensional images. There was an increase in bone volume, trabecular number, and trabecular thickness with a concomitant decrease in trabecular spacing. Analysis of a nonengrafted mouse or a mouse that was transplanted with BM cells from oim mice showed continued deterioration in the bone parameters. The engrafted mice gained weight and became less prone to spontaneous fractures while the control mice worsened clinically and eventually developed kyphosis. CONCLUSIONS These findings strongly support the concept that HSCs generate bone cells. Furthermore, they are consistent with observations from clinical transplantation studies and suggest therapeutic potentials of HSCs in OI.